Intratumor genetic heterogeneity in squamous cell carcinoma of the oral cavity

BackgroundWe sought to evaluate intratumor heterogeneity in squamous cell carcinoma of the oral cavity (OCC) and specifically determine the effect of physical separation and histologic differentiation within the same tumor.MethodsWe performed whole exome sequencing on five biopsy sites—two from well‐differentiated, two from poorly differentiated regions, and one from normal parenchyma—from five primary OCC specimens.ResultsWe found high levels of intratumor heterogeneity and, in four primary tumors, identified only 0 to 2 identical mutations in all subsites. We found that the heterogeneity inversely correlated with physical separation and that pairs of well‐differentiated samples were more similar to each other than analogous poorly differentiated specimens. Only TP53 mutations, but not other purported “driver mutations” in head and neck squamous cell carcinoma, were found in multiple biopsy sites.ConclusionThese data highlight the challenges to characterization of the mutational landscape of OCC with single site biopsy and have implications for personalized medicine.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150549/1/hed25719.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150549/2/hed25719_am.pd

TNF recruits TRADD to the plasma membrane but not the trans-Golgi network, the principal subcellular location of TNF-R1

The subcellular localization of TNF-R1 to the Golgi apparatus, initially observed in endothelial cells, has been confirmed using transfection of bovine aortic endothelial cells with a human TNF-R1 expression plasmid, The subcellular interactions of TNF-RI and the TRADD (TNFR-associated death domain protein) adaptor protein have been analyzed in the human monocyte cell line U937 and the human endothelial cell line ECV304 by confocal immunofluorescence microscopy and by Western blot analysis of fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but principally localizes to the trans-Golgi network, TRADD is concentrated in the cis- or medial-Golgi region, but separates from the Golgi during cell fractionation, Coimmunoprecipitation studies have shown that TRADD binds to TNF-R1 within 1 min of TNF treatment in a cell fraction-containing plasma membrane. This association is followed by a gradual dissociation, which is prevented if receptor-mediated endocytosis is inhibited by hypertonic medium. In contrast, no association is detected between TRADD and TNF-R1 in the Golgi in response to exogenous TNF at any time examined, These results suggest that although TNF-R1 is predominantly a Golgi-associated protein and TRADD also localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF-R1 only at the plasma membrane

Tumor necrosis factor is delivered to mitochondria where a tumor necrosis factor-binding protein is localized

The roles of the known tumor necrosis factor (TNF) receptors (TNFR-I and TNFR-II) and their associated signaling pathways in mediating the diverse actions of TNF remain incompletely defined. We have found that a proportion of exogenous TNF is delivered to mitochondria as well as to lysosomes. Using confocal and immunoelectron microscopy and Western blotting of subcellular fractions, we have identified a 60-kd protein in the inner mitochondrial membrane that is recognized by a monoclonal antibody to TNFR-II. In isolated mitochondria, this protein binds [I-125]-TNF. This provides evidence of a mitochondrial binding protein for an extracellular ligand and demonstrates the presence of a pathway capable of delivering TNF from the cell surface to mitochondria. These findings suggest that TNF effects on cells may be due in part to a direct effect on mitochondria

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins

TNF and TNF receptor expression and insulin sensitivity in human omental and subcutaneous adipose tissue--influence of BMI and adipose distribution

Tumour necrosis factor (TNF)alpha is implicated in the relationship between obesity and insulin resistance/ type 2 diabetes. In an effort to understand this association better we (i) profiled gene expression patterns of TNF, TNFR1 and TNFR2 and (ii) investigated the effects of TNF on glucose uptake in isolated adipocytes and adipose tissue explants from omental and subcutaneous depots from lean, overweight and obese individuals. TNF expression correlated with expression of TNFR2, but not TNFR1, and TNF and TNFR2 expression increased in obesity. TNFR1 expression was higher in omental than in subcutaneous adipocytes. Expression levels of TNF or either receptor did not differ between adipocytes from individuals with central and peripheral obesity. TNF only suppressed glucose uptake in insulin-stimulated subcutaneous tissue and this suppression was only observed in tissue from lean subjects. These data support a relationship between the TNF system and body mass index (BMI), but not fat distribution, and suggest depot specificity of the TNF effect on glucose uptake. Furthermore, adipose tissue from obese subjects already appears insulin 'resistant' and this may be a result of the increased TNF levels

TNF-α-Induced Mitochondrial Alterations in Human T Cells Requires FADD and Caspase-8 Activation but Not RIP and Caspase-3 Activation

Although much is known about how TNF-α induces apoptosis in the presence of inhibitors of protein synthesis, little is known about how it induces apoptosis without these inhibitors. In this report we investigated temporal sequence of events induced by TNF-α in the absence of protein synthesis. Regardless of whether we measured the effects by plasma membrane phosphotidylserine accumulation, by DNA strand breaks, or activation of caspases, significant changes were observed only between 12–24 h of TNF-α treatment. One of the earliest changes observed after TNF-α treatment was mitochondrial swelling at 10 min; followed by cytochrome c and Smac release at 10–30 min, and then heterochromatin clumping occurred at 60 min. While genetic deletion of receptor-interaction protein (RIP) had no effect on TNF-α-induced mitochondrial damage, deletion of Fas-associated death domain (FADD) abolished the TNF-induced mitochondrial swelling. Since pan-caspase inhibitor z-VAD-fmk abolished the TNF-α-induced mitochondrial changes, z-DEVD-fmk, an inhibitor of caspase-3 had no effect, suggesting that TNF-α-induced mitochondrial changes or cytochrome c and Smac release requires caspase-8 but not caspase-3 activation. Overall, our results indicated that mitochondrial changes are early events in TNF-α-induced apoptosis and that these mitochondrial changes require recruitment of FADD and caspase-8 activation, but not caspase-3 activation or RIP recruitment. Antioxid. Redox Signal. 13, 821–831